A variety of methods have been developed to facilitate the transfer of genetic material into specific cells. These methods are useful for both in vivo or ex vivo gene transfer. In the former, a gene is directly introduced (intravenously, intraperitoneally, aerosol, etc.) into a subject. In ex vivo (or in vitro) gene transfer, the gene is introduced into cells after removal of the cells from specific tissue of an individual. The transfected cells are then introduced back into the subject.
Delivery systems for achieving in vivo and ex vivo gene therapy include viral vectors, such as retroviral vectors or adenovirus vectors, microinjection, electroporation, protoplast fusion, calcium phosphate, and liposomes (Felgner, J., et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987); Mulligan, R. S., Science 260:926-932 (1993); Morishita, R., et al., J. Clin. Invest. 91:2580-2585 (1993)).
Delivery of genetic material to cells using liposomal carriers has been widely studied. It is generally understood that liposome vesicles are taken up by cells via endocytosis and enter the lysosomal degradation pathway. Thus, some effort towards designing liposomes that avoid degradation has been made.
The use of cationic lipids, e.g., derivatives of glycolipids with a positively charged ammonium or sulfonium ion-containing headgroup, for delivery of negatively-charged biomolecules, such as oligonucleotides and DNA fragments, as a liposome lipid bilayer component is widely reported. The positively-charged headgroup of the lipid interacts with the negatively-charged cell surface, facilitating contact and delivery of the biomolecule to the cell. The positive charge of the cationic lipid is further important for nucleic acid complexation.
However, systemic administration of such cationic liposome/nucleic acid complexes leads to their facile entrapment in the lung. This lung localization is caused by the strong positive surface charge of the conventional cationic complexes. In vivo gene expression of the conventional cationic complexes with reporter gene has been documented in the lung, heart, liver, kidney, and spleen following intravenous administration. However, morphological examination indicates that the majority of the expression is in endothelial cells lining the blood vessels in the lung. A potential explanation for this observation is that the lung is the first organ that cationic liposome/nucleic acid complexes encounter after intravenous injection. Additionally, there is a large surface area of endothelial cells in the lung, which provides a readily accessible target for the cationic liposome/nucleic acid complexes.
Although early results were encouraging, intravenous injection of simple cationic liposomes has not proved useful for the delivery of genes to systemic sites of disease (such as solid tumors other than lung tumors) or to the desired sites for clinically relevant gene expression (such as p53 or HSV-tk) Cationic liposomes are cleared too rapidly, and present a host of safety concerns.
Another approach has been to include in the liposome a pH sensitive lipid, such as palmitoylhomocysteine (Connor, J., et al., Proc. Natl. Acad. Sci. USA 81:1715 (1984); Chu, C.-J. and Szoka, F., J. Liposome Res. 4(1):361 (1994)). Such pH sensitive lipids at neutral pH are negatively charged and are stably incorporated into the liposome lipid bilayers. However, at weakly acidic pH (pH<6.8) the lipid becomes neutral in charge and changes in structure sufficiently to destabilize the liposome bilayers. The lipid when incorporated into a liposome that has been taken into an endosome, where the pH is reported to be between 5.0-6.0, destabilizes and causes a release of the liposome contents.
In addition, tumor cell direct targeting is much more challenging than angiogenic endothelial cell targeting. Liposome/DNA complexes access angiogenic endothelial cells of tumor vasculature relatively easily, since the cells are directly exposed in the blood compartment. For targeting of tumor cells, liposome/DNA complexes need to extravasate through the leaky tumor blood vessels and then can reach tumor cells. Thus the complex stability, size, surface charge, blood circulation time, and transfection efficiency of complexes are all factors for tumor cell transfection and expression.